Preparing biological samples for analysis

ABSTRACT

Methods and devices for preparing a biological sample for analysis are described. The biological sample from an organism has at least macromolecule having a primary structure that naturally degrades after the sample is removed from the organism. The method includes causing the biological sample to adopt a shape to permit rapid and uniform heating. The shaped sample is then rapidly and uniformly heated, thereby altering a secondary structure of the macromolecule while preserving its primary structure.

CLAIM OF PRIORITY

This application is a continuation-in-part of, and claims the benefit ofpriority under 35 U.S.C. §120 to, U.S. patent application Ser. No.11/212,454, filed Aug. 26, 2005 now abandoned, which published as U.S.Publication No. 20070048877 on Mar. 1, 2007, and which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to methods and apparatus for preparing abiological sample for analysis. The invention more particularly relatesto methods and apparatus for heating a biological sample soon after itis extracted so that the primary structures of proteins in it are notdegraded.

BACKGROUND

It is key to any analysis of a biological sample that the integrity ofits constituents is conserved between the time that the sample isextracted from a living organism and the time that analysis is carriedout. Sample degradation, however, is both hard to impede, and hard todetect. The result is that many analyses miss the presence of speciesthat have degraded long before the analysis is carried out;correspondingly, such analyses may in fact identify degradation productsof critical components in place of the original components.

Since the sequencing of the human genome and the realization that theremay be far fewer genes than was originally thought, attention has turnedto the proteome; it is now believed that it is the assemblage ofproteins in an organism that is the key to understanding physiology,disease, and function. Proteins are found in many differentenvironments, for example, in cell nuclei, organelles, protoplasm, andmembranes, as well as the inter-cellular space, and in body fluids suchas blood. Despite their ubiquity, proteins are extremely sensitive totheir environments and thus are not always easy to detect and toidentify because they can degrade very quickly.

A protein is composed of one or more strings (polypeptide chains) of theresidues of the 20 naturally occurring amino acids, which fold intospecific 2- and 3-dimensional structures that determine the protein'sactivity. A given protein has a unique sequence of amino acids, termedits primary structure. The secondary structure is defined by dihedralangles (referred to as phi and psi) of the backbone atoms of the aminoacid residues, and the hydrogen bonds between side chain and backboneatoms. The dihedral angles and patterns of hydrogen bonds within certaincharacteristic subsequences of consecutive (and non-consecutive)residues, can give rise to units of secondary structure that arerelatively stable, e.g., so-called alpha helices, and beta sheets.

The tertiary structure of a protein is the term used to refer to how thesecondary structure units and the polypeptide chains that connect themfold into a three dimensional structure. The quaternary structure refersto how two or more non-contiguous polypeptide chains that each adopttheir own tertiary structure also associate with one another to form aprotein. A protein's function may derive from either or both of itstertiary and quaternary structure. Typically the three-dimensionalconformations adopted by the one or more polypeptide chains give rise tofeatures, often described as clefts, cavities, or grooves depending ontheir geometry, that can bind to other molecules with high specificity.Such other molecules include drugs, nucleic acids, and mostsignificantly for sample integrity, other proteins and polypeptides.

The natural functions of the assemblage of proteins in an organism arekept in check by a complex but delicate balance of biochemical pathwayswhile the organism is alive. Once an organism dies, or once a sample oftissue is extracted from a living organism, the regulatory balance ofthe organism or in the sample is lost and key proteins start to breakdown. The breakdown can manifest itself in a number of different ways.For example, some proteins whose natural role is to digest otherproteins (a “proteolytic” function), and whose natural levels are keptin check while an organism is alive, may go out of control after death.Thus, many proteins and key polypeptides such as coactivators, hormones,and corepressors, end up being actually digested by naturally occurringproteolytic proteins in the sample. Digestion typically involves arupturing of the polypeptide backbone at one or more points, therebyresulting in protein or peptide fragments. Still other proteins maynaturally decompose by other means, such as hydrolysis; whereas in aliving organism their levels are maintained because they are continuallysynthesized, after death they rapidly disappear. For example,post-mortem activity of proteases and oxidative stress has been shown toplay an important role on peptide and protein concentration in thebrain, as well as for detecting post-translational modifications (K.Sköld et al., “A Neuroproteomoic Approach to Targeting Neuropeptides inthe Brain”, Proteomics, 2, 447-454, (2002); M. Svensson et al.,“Peptidomics-Based Discovery of Novel Neuropeptides”, J. Proteome Res.,2, 213-219, (2003), both of which are incorporated herein by reference.

For purposes of protein identification, however, to determine whatproteins are present in a sample, it is sufficient to be able toascertain their respective primary structures, i.e., sequences. Proteinsand polypeptides have been widely investigated by methods such as twodimensional gels and mass spectrometry, but such techniques depend onhaving access to samples in which natural protein degradation has notadvanced to a point where the concentrations of critical species havebeen reduced below the various measurement thresholds.

To study proteins and peptides, tissue or cell samples are usuallydisrupted by homogenization in certain specific buffer conditions. Thesebuffers often contain ingredients that are supposed to cause a cessationof all protein activity, including proteins (proteases) that degradeother proteins. However, the study of tissue samples from patients ormodel organisms usually exposes the samples to a certain period ofoxygen and nutrient depletion before homogenization and proteaseinactivation occurs.

Consequently, techniques have been developed in the art for attemptingto preserve biological samples after extraction and prior to analysis.Examples of such techniques include tissue fixation, which typicallyinvolves immersing a sample in an aldehyde solution, and irradiatingsamples with microwaves (see, e.g., Theodorsson, et al., “MicrowaveIrradiation Increases Recovery of Neuropeptides From Brain Tissues”,Peptides, 11:1191-1197, (1990)). Use of aldehyde solutions isproblematic because it doesn't arrest natural degradation of proteins(though it is somewhat effective at maintaining large-scale structure oftissues). Microwave irradiation is problematic because it is generallynon-uniform, that is, some parts of the sample reach a temperature thatis high enough to cause sample breakdown. (See, for example, Fricker etal., “Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brainand Pituitary”, Molecular & Cellular Proteomics, 4:1391-1405, (2005).)Furthermore, microwave irradiation has formerly been applied to living(non-human) subjects as part of a sacrificial protocol and thus has yetto be established as a tool for analyzing samples that have beenextracted from subjects, both human and non-human.

Accordingly, there is a need for a reliable technique for preserving thecontents of tissue samples prior to analysis in a way that impedesnatural degradation and that acts on a given sample reliably.

The discussion of the background to the invention herein is included toexplain the context of the invention. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge as at the priority date of any ofthe claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The present invention comprises a method for preparing a biologicalsample for analysis. The biological sample comprises at least oneprotein or polypeptide, having an amino acid sequence, from an organism.The method comprises causing a volume of the sample to adopt a shapewherein the shape permits uniform and rapid heating, thereby forming ashaped sample; and heating the shaped sample so that the secondarystructure of the macromolecule is disrupted, but the primary structureis not. In an embodiment, the biological sample is given a shape thatfacilitates an effective heating in terms of the heating being uniformand fast. This helps to shorten the time needed to reach a disruptedsecondary structure. By blocking certain biological processes driven byproteins, degradation of other constituents of the sample is avoided.Because the time between taking the biological sample and performing abiological analysis has a large impact on the level of degradation, evenafter a short time, e.g., after as little as 1-3 minutes, it isimportant that heating takes place immediately after taking the sample.By heating the tissue proteins that function as proteases, theirsecondary and tertiary structure, and thereby their function, is lost.

The method preserves the primary structure of proteins and peptides butsimultaneously disrupts their original secondary, tertiary structures,and, where applicable, quaternary structures. The heating of the sampletherefore has several advantages, including enabling species such as therelatively low-abundant neuropeptides and proteins that would otherwisebe digested to remain intact. In addition, the method minimizesdegradation of neuropeptides and proteins in a reproducible manner. Thismethod also makes it possible to compare the content and levels ofproteins and peptides from different samples. Also, because a sample canbe taken from an organism without sacrificing the entire organism, themethod may be non-fatal, i.e., the organism does not have to perish as aconsequence of using the method.

The present invention further includes a method for preparing abiological sample for analysis, the sample comprising at least onemacromolecule having a primary structure and a secondary structure, themethod comprising: causing a volume of the sample to adopt a shapewherein the shape permits uniform and rapid heating, thereby forming ashaped sample; and heating the shaped sample so that the secondarystructure of the macromolecule is disrupted, but the primary structureis not, wherein the heating reduces proteolytic activity by at least70%.

The present invention still further includes a method for preparing abiological sample for analysis, the sample comprising a proteolyticmolecule and a polypeptide, wherein the polypeptide have a primarystructure and secondary structure, the method comprising: heating thebiological sample to cause the sample to uniformly attain a temperatureat which the activity of the proteolytic molecule is disrupted enough sothat the proteolytic molecule are unable to degrade the primarystructure of the polypeptide.

The present invention also includes a method for preparing a biologicalsample from an organism for analysis, the sample comprising at least onemacromolecule of interest, wherein the macromolecule of interest has aprimary structure and a secondary structure, the method comprising:causing a volume of the sample to adopt a shape wherein the shapepermits rapid and uniform heating, thereby creating a shaped sample; andheating the shaped sample to cause the shaped sample to attain atemperature wherein the temperature causes a secondary structure of adigestive molecule to degrade, wherein the digestive molecule naturallydigests the macromolecule of interest when its secondary structure isintact, and wherein the temperature does not cause the primary structureof the macromolecule of interest to degrade.

The present invention even further includes a system for preparing abiological sample, the system comprising: a heat source; a retainingmember in communication with the heat source, and configured to contactthe biological sample, and wherein the retaining member conducts heatfrom the heat source into the biological sample; a zone in which thebiological sample can be held at a controlled temperature; and atransfer element configured to move the biological sample out from thezone and onto the retaining member.

The present invention yet further includes a heating device, comprising:a chamber configured to receive a shaped biological sample, wherein thechamber has one or more internal surfaces that are in contact with thebiological sample, and wherein the sample is totally contained withinthe chamber; one or more heating elements in communication with the oneor more internal surfaces; a heat sensor in communication with the oneor more internal surfaces; an inlet adapted to permit the sample to bedirected into the chamber; and wherein the chamber has a shape so thatno part of the sample is greater than 10 mm from a point on any one ofthe one or more internal surfaces.

The present invention additionally includes a fixed treated biologicalsample, comprising: a sample of biological material that has beenextracted from an organism and that has a macromolecule which is notdegraded, at least 60% of which has been denatured, per unit volume, ascompared to the same macromolecule in the biological material in vivo.

DESCRIPTION OF DRAWINGS

FIG. 1 shows charts of sample quality, post sampling.

FIG. 2 shows a flow chart of a method according to the invention.

FIG. 3 shows schematics of four exemplary shapes of biological samplesfor use with the invention.

FIG. 4 is a schematic of a device for preparing a biological sample foranalysis.

FIGS. 5-8 are schematics of devices for preparing a biological samplefor analysis.

FIG. 9 is a perspective view of a device for preparing a biologicalsample for analysis.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION Overview

The present invention involves methods and apparatus for preparing abiological sample for analysis. The sample, which has been extractedfrom an organism, contains various macromolecules such as polypeptidesor proteins. In order to prepare the sample for analysis, the naturaldegradation of the primary structures of the various macromolecules isarrested to the fullest extent possible.

Accordingly, in one embodiment, after a first period of time which isone over which degradation is minimal, or preferably kept at or aroundlevels similar to those found in the sample when in vivo, the sample iscaused to adopt a shape that permits rapid and uniform heating. Then,the sample is rapidly and uniformly heated over a second period of time,and in such a manner that all parts of the sample attain a particulartemperature. The temperature is referred to as the denaturationtemperature because it is a temperature at which various macromoleculesdenature, i.e., their secondary, tertiary, and/or quaternary structureis disrupted, but it is not a temperature at which the primary structureof the macromolecules is degraded. Preferably the macromolecules thatare denatured include at least those macromolecules that play a role inthe natural degradation processes of the sample. For example, suchmacromolecules include proteolytic enzymes that, if not denatured, woulddegrade—e.g., by digesting—other molecules in the sample.

It is to be understood that the conditions deployed herein lead to aneffect of degree, rather than one that is absolute: it is of courseunderstood that no chemical reaction can be halted altogether (save atthe practically unattainable Absolute Zero of temperature). Thus, theeffect of increasing (or decreasing) temperature, typically expressedmathematically by the exponential Arrhenius relationship, is one inwhich a statistically greater number of molecules react in a given wayper second, as temperature is increased. Thus it is to be understoodthat, for example, although the temperatures employed are chosen to behigh enough to cause denaturation, but low enough not to causedegradation of primary structure of a given set of molecules, it doesnot mean that a small number of those molecules do not still undergodegradation under those conditions. It is sufficient for the purposes ofthe present invention that the number of such molecules is insignificantand is, for example, less than 5% of the population of initialmolecules, and is preferably less than 2%, and even more preferably lessthan 1%, and still more preferably less than 0.1% of the initialpopulation of those molecules.

It is also to be understood that, when the term secondary structure isused herein, it can mean the overall three-dimensional configuration ofa macromolecule that is responsible for its activity and specificity.Thus, the term secondary structure can mean, herein, features of amacromolecule that are commonly referred to distinctly as secondary,tertiary, and quaternary structure.

In a second embodiment, the sample is not intended to be analyzed soonafter extraction but instead is intended to be stored prior to analysis.In such an embodiment, the sample is frozen as soon as is practicallypossible after extraction. The time between extraction and the time whenthe sample attains a frozen temperature is one over which degradation isminimal. The sample is caused to adopt a shape that permits rapid anduniform heating. The sample can also be caused to adopt such a shapewhile frozen, or prior to freezing. Then, the sample is rapidly anduniformly heated over a second period of time, and in such a manner thatall parts of the sample attain the denaturation temperature. It is to beunderstood that the second period of time for a frozen sample is notnecessarily the same as the second period of time for a sample that hasnot been frozen prior to heating. Preferably the second period of timeis rapid so that the sample does not undergo a period of thawing inbetween its frozen state and the heated state. For example, rapidheating preferably occurs over less than a minute, less than 30 seconds,less than 20 seconds, less than 10 seconds, less than 5 seconds, or lessthan 2 seconds.

The present invention has contemplated application to analysis ofpolypeptides, proteins (including antibodies), carbohydrates, lipids,hormones, and metabolites in a biological sample. It would beunderstood, however, that study of other molecules and macromoleculesmay also benefit from the methods and apparatus described herein. Forexample, and in particular, any other macromolecules in a biologicalsample that have a three-dimensional conformation that may be disruptedby heating while preserving the sequence of chemical bonds within themcan be preserved for analysis by the methods and apparatus describedherein. Such other macromolecules include, but are not limited to,nucleic acids and oligonucleotides. Macromolecules are understood,generally, to be molecules of high molecular weight that are composed ofrepeating units of same or different identities. Similarly the methodsof the present invention may also lead to more accurate detection ofsmall molecules (non-macromolecules) that would otherwise be digested ordegraded by other means.

The terms disrupted and degraded are used herein to refer to alterationof molecular structures in a sample. A structure is disrupted if it isaltered in such a way as to impair its function, even though thestructure's identity is not destroyed. Thus, a protein, for example, canbe denatured and, in so doing, its secondary, and/or tertiary and/orquaternary structure is disrupted, i.e., altered such as by unraveling,so that its function is destroyed. However, such a process does notchange its primary sequence and thus its identity is maintained.Conversely, a structure is degraded if its chemical identity is changed.Thus, for example, cleaving a protein to produce two or more fragmentshas degraded the protein because not only has its secondary and/or itstertiary and/or its quaternary structure been altered, but its primarystructure has too.

The samples for use with the present invention may comprise anybiological sample from an organism. Thus, the samples include, but arenot limited to, tissue, muscle, bone, bone marrow, tooth, hair, skin, orany organ such as brain, kidney, liver, stomach, intestine, reproductiveorgans, or pancreas. The samples further include body fluids including,but not limited to tears, saliva, blood, semen, sweat, or urine.

The organism is preferably a mammal, but may be a reptile, aninvertebrate, a fish, an insect, or a bird. The organism is still morepreferably a human, but may be an animal, including, but not limited to:a non-human primate, rabbit, sheep, dog, cat, horse, monkey, mouse, orrat.

Exemplary Theory

While a tissue is living, proteins are synthesized and degraded. This isa dynamic process and is extensively controlled by various mechanisms.For example, proteolysis naturally occurs within living tissue but it istypically regulated so that proteins that are proteolyzed remain insufficient quantities to perform their functions. A disease state canchange this balance, and hence, a change in the balance can be used tocharacterize a disease.

The peptidome of a sample, the set of peptides present in a specificcell, tissue, organism or system, is directly linked to its proteome.The distribution of molecules between proteome and peptidome iscontrolled by proteases and protease inhibitors. Post-mortem enzymeactivity plays a role in the integrity of the peptide and proteincontent in tissues, such as brain tissue. There is always a low level ofhighly abundant peptides from protein degradation in a sample thatarises from the natural protein-peptide homeostasis.

Many studies of tissue and cells requires their removal from thesupportive environment of a living organism, thus disturbing the variousregulatory processes, and in particular leading to deprivation of oxygenand nutrients in the sample, for example as blood flow to the tissue ishalted. Ischemia, a restriction on blood delivery to tissue, leads tosubsequent hypoxia and anoxia.

The degradation of tissue has been particularly closely studied forsamples of brain tissue. Thus, even though brain cells do not contain areservoir of oxygen in the same way that muscle cells do, i.e., withmyglobin, their rate of oxygen utilization is high. To ensure brain cellsurvival, a constant supply of oxygen and nutrients are required. Adilemma in the study of brain tissue is that, with many types ofanalytical techniques, the brain tissue must be removed from its oxygenand nutrient supplying environment. Without oxygen, oxidativephosphorylation and subsequent adenosine triphosphate (“ATP”) productionis halted, causing deficiencies in cell functions. The time at whichdegradation of brain tissue begins is much shorter than the time fordegradation of other biological tissues or bodily fluids. Furthermore,even within the brain, the protein and polypeptide degradation time isnot uniform. Oxygen retention is generally low and non-uniform withlarge variation between different brain structures. It is generallyhigher in the regions rich in cell bodies and dendrites, such as thegrey matter of the cortex, and lower in areas where fibers predominate,such as the white matter of the cortex, pons, and fornix (see, e.g.,Erecinska, et al., “Tissue Oxygen Tension and Brain Sensitivity toHypoxia”, Respir. Physiol., 128, 3:263-276, (2001)).

Glucose is the main metabolic substrate for the adult brain. Glucose ismetabolized through glycolysis to pyruvate, which enters the Krebs cyclein mitochondria where, in the presence of oxygen, it is completelyoxidized to carbon dioxide and water (see, e.g., Goldman, et al.,“Acid-induced Death in Neurons and Glia” J. Neuroscience, 11:2489-2497,(1991)). A decrease of oxygen interferes with the oxidation of pyruvatein the mitochondria. As a result, mitochondrial ATP production iscompromised, leaving only glycolytic ATP production. In the ischemicbrain, ATP generation occurs via anaerobic conversion of endogenoussubstances.

As noted, the brain contains only a paucity of oxygen stores. The storesof oxygen in blood vessels can support normal oxygen consumption in thebrain for only a few seconds. Anaerobic glycolysis only yields 2 mol ofATP per mol of glucose, as compared to ˜35 mol of ATP under aerobicglycolysis. This results in a utilization of endogenous stores of ATP,ADP and phosphocreatine (PCr). Creatine phosphate donates a phosphorgroup to ADP, thereby converting it to ATP. The high-energy phosphatecompounds including creatine phosphate, are present in vitro in bothneurons and glia at comparable concentrations. (See, e.g., Folbergrova,et al., “Phosphorylase Alpha and Labile Metabolites During Anoxia:Correlation to Membrane Fluxes of K⁺ and Ca²⁺ ”, J. Neurochem., 55(5):1690-6, (1990)).

By using these endogenous energy substrates, energy metabolism can besupported for approximately one minute in ischemia (Hansen, A. J.,“Effect of Anoxia on Ion Distribution in the Brain”, Physiol. Rev., 65,1:101-48 (1985).). In ischemia studies, glucose levels are depleted andlactate levels are 3-fold increased after 60 seconds. After 2 minutes,lactate levels are increased 5-fold (Folbergrova, et al.). Theutilization of high-energy phosphate groups is reduced to 30% afterapproximately 10 seconds, 15% after the first minute, and to nearly zeroafter 2 minutes (Hansen; Folbergrova, et al.).

There is an efflux of K⁺ ions from rat brain cortex immediately afterinduction of anoxia through cardiac arrest (Hansen). There is a slowincrease of K⁺ ions during the first two minutes of anoxia (K-phase I).After about 2 minutes, the extracellular K⁺ ion concentration rises from10 mM to about 60 mM within a few seconds (K-phase II). The rapidincrease in extracellular potassium takes place when the ATP energymetabolism and oxygen consumption have fallen to very low levels;between 1 and 2 minutes after ischemia. During the next few minutes theextracellular K⁺ levels rises slowly to 80 mM (K-phase III). The slowrise during K-phase I may be due to insufficient inward pumping of K⁺ions due to reduced N—-K-ATPase activity. After 1 to 2 minutes ofischemia, ATP energy levels are insufficient to support Na—K-ATPaseactivity, causing depolarisation and a reduction of Na, K, Ca, and Cl(see, e.g., Hille, B., Ionic Channels in Excitable Membranes, SinauerAssociations, Sunderland, Mass., (1992)).

Complete ischemia in rat cortex induces a rapid increase inintracellular Ca levels after approximately 60 seconds (Kristian, T.,“Metabolic Stages, Mitochondria and Calcium in Hypoxic/Ischemic BrainDamage”, Cell Calcium, 36, 3-4:221-33, (2004)). The ischemia-inducedchanges in ion homeostasis causes a depolarization, causing entry of Cathrough voltage-dependent Ca-levels and NMDA-receptors. NMDA antagonisttreatment of ischemic rat cortex delays the intracellular Ca increasewithin 30 seconds (Kristian). The Ca-ATPase activity and theCa-sequestration into organelles is ATP driven by and thereforesensitive to the rate of energy metabolism. Increased glucose levels inrat cortex also delays Ca influx to 90 seconds (Kristian). When theenergy metabolism is compromised, Ca is released from the organelles.The increase of Ca can activate K—Ca channels, thereby promoting Kefflux (Hille). As the ion balance is lost, the cell organellescollapses and proteins and peptides are released and degraded.

As described, after removing a sample of tissue from a living organism,proteolysis increases or can continue unchecked, thereby rapidly leadingto destruction of proteins and polypeptides within the sample.Therefore, to obtain the most information about the tissue's protein andpolypeptide makeup, the sample should be heated as quickly as possibleafter removing the sample from the organism. Without being constrainedto any particular theory, it is believed that the tissue should beheated prior to the ATP levels dropping below a point that ion gradientsare no longer maintained in the cells in the sample. The electrochemicalgradient across a cell membrane, manifested by concentration gradientsof ions such as sodium and potassium, provides a source of energy forintra-cellular chemistry. Enzymes such as Na⁺ ATPase and K⁺ ATPase useATP to create and maintain such gradients. Once a cell experiencesenergy failure, that is, once the ATP level drops below a thresholdlevel, calcium accumulates in the intracellular space as a result of thedisturbed ion homeostasis. As the ion balance is lost, the cellorganelles collapses and proteins and peptides are released anddegraded.

The degradation of exemplary mouse brain tissue is described withrespect to the changes the sample undergoes after the sample isextracted from an organism, in FIG. 1. After 15 seconds have elapsedfrom the time that the tissue is removed from an organism, there isabout 25-50% less ATP, 50% less glucose and 50% more lactate in thetissue than in a similar sample of tissue that is still in the organism.After 45 seconds, there is about 75% less glucose and 150% more lactate.After 1 minute, there is about 50% less ATP, the pH has decreased(signifying increased acidity in the sample), the glucose is gone, thereis about 200% more lactate, the sodium/potassium ATPase stopsfunctioning, potassium depolarization occurs, and cytosolic calciumincreases. After 2 minutes, there is typically no ATP remaining, thereis about 350% more lactate, calcium activated protease increases andphosphorylation no longer occurs. After 3 minutes, the proteins degrade.Thereafter, necrosis (when supplies of oxygen are cut off) or apoptosisoccurs. Stathmin, a 17 kDa protein, has been suggested as a marker forprotein degradation. A mouse brain was analyzed for fragments ofstathmin. At times between 0 and 1 minute, stathmin fragments are verylow, shown as less than 5,000 units of ion intensity as detected by amass spectrometer (***P<0.0001, ANOVA, t-test). After three minutes, onaverage, the stathmin fragments increase dramatically, which isindicated by the ion intensity of more than 600,000. After 10 minutesfrom extracting the sample, the stathmin fragments increase again, whichis indicated by an ion intensity just under 1,000,000. Determining thedegradation of stathmin is described further in “Method for Determiningthe Quality of a Biological Sample”, U.S. Provisional Patent ApplicationNo. 60/740,542, filed Nov. 29, 2005, incorporated herein by reference.

Accordingly, in certain embodiments, an indication of sample degradationis obtained by measuring the amount of stathmin fragments in the sample,after it has been prepared by the methods described herein. If thelevels of stathmin fragments are substantially higher in the sample thantheir corresponding levels in vivo, or if the ratio between the Sthaminprotein and its degradation product is much higher than it is in vivo,i.e., if C(Stathmin)_(in vivo)/C(Stathminfragment)_(in vivo)>>C(Stathmin)_(ex vivo)/C(Stathminfragments)_(ex vivo), then too much sample degradation has taken place.Preferably, the difference between the ratios measured in vivo and exvivo does not exceed 50% (i.e., (Ratio_(in vivo)/Ratio_(ex vivo))<1.5),and still more preferably does not exceed 40%, and even more preferablydoes not exceed 30%, and yet more preferably does not exceed 20%, andmost preferably does not exceed 10%.

For these reasons, a biological sample that is prepared using themethods described herein is denatured prior to the ion balance withinthe sample becoming sufficiently imbalanced (as compared to a similarsample in a living organism) that the level of molecular fragments, suchas peptide fragments from proteins, found in the analyzed sample arecomparable to the levels in a similar sample found in a living organism.Because ion imbalance occurs after the ATP level drops, a denaturedsample may have some amount of ATP remaining.

Process

Steps of a method for preparing a biological sample for subsequentanalysis according to the present invention are shown in FIG. 2. Thebiological sample comprises at least one protein or polypeptide, havingan amino acid sequence, from an organism. The biological sample isextracted 2 from the organism. The sample is optionally frozen 4. Then,after a first period of time, the sample is caused to adopt a shape 6such that the shape permits uniform and rapid heating. After the samplehas been so shaped, the shaped sample is then heated 8 for a secondperiod of time such that all portions of the sample are heated atrelatively the same rate and achieve the same temperature atapproximately the same time. Throughout the causing and heating, thesample is handled so that a minimal amount of degradation of the primarystructure of the at least one polypeptide or protein occurs prior to thesample being heated. Finally, the sample may be subjected to furtheranalysis.

Extraction

Extraction of a sample from an organism may take a number of forms. Forexample, the sample may be excised from the organism by cutting, takinga smear, or by drawing out with a syringe or a catheter.

While generally all biological samples undergo similar steps thateventually lead to necrosis, wherein ATP production and phosphorylationis halted, ion gradients are lost, the cell organelles collapse,proteins and peptides are released, and proteolysis increases, the rateof each of these steps depends at least in part on the type of sample.Thus, although massive degradation does not occur in some samples untilas much as 10 minutes have elapsed, in some samples, massive degradationcan occur as quickly as 3 minutes, 2 minutes, 1 minute, 30 seconds, 10seconds or less from the time the tissue is removed from the organism.Accordingly, the period of time between extraction of a sample from anorganism and the time that the sample is heated (as further describedherein) is preferably 3 minutes, still more preferably 2 minutes, andeven more preferably between 10 seconds and 2 minutes. In someembodiments, to avoid degradation of the sample, the sample can beextracted by an instrument that simultaneously removes the sample andshapes the sample into the desired shape. The sample is then immediatelyintroduced into the heating device to arrest proteolysis.

Sample Shape

Determining how to shape the sample so that the sample can be uniformlyheated preferably takes into account the type of device to be used toheat the sample, and various characteristics of the sample. The samplecan be heated by one or more of the well-known forms of heat transfer:conduction, convection or radiation. If the sample is heated byconduction heating, a factor in determining how to choose the shape ofthe sample is that it is preferable that no part of the sample interioris greater than a threshold distance from a source of heat. Preferablythis threshold is 5 mm, though it may vary with the nature of the tissuesample. For example, it may be 1 mm, 2 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm,5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 9 mm, or 1 cm. In someembodiments, it is preferable that the sample is shaped to have thelargest surface area to volume ratio possible, such as by creating avery thin slice, preferably one that is uniformly thin. Where theheating device has conductive elements that are in specific shapes, suchas a cylindrical heating element or a probe which is inserted into thesample, a cylindrical shape for the sample may be more desirable. Theshape of the sample should be one that maximizes any temperaturegradient that occurs across the sample during heating so that the heatconduction from the surface to the interior of the sample is aseffective as possible. The speed of the heating step can be kept at arate sufficiently fast, but also be selected to prevent the temperaturefrom going too high, or from some parts of the sample from being heatedtoo slowly and not being heated to the same temperature as the rest ofthe sample. If part of the sample is allowed to go beyond a maximumtemperature, water in the sample may boil and the cell structure may bedestroyed. In more extreme instances, the primary structure of a proteinor polypeptide of interest may be destroyed by temperatures that are toohigh. Conversely, if part of the sample does not reach the denaturationtemperature, the whole sample can be tainted by residual presence of theun-denatured portion. Uniform heating avoids both of these outcomes.

Referring to FIG. 3, four embodiments of shapes of biological samplessuitable for use with the present invention are shown. Parallelepipeds1, 3 and a right cylinder 5 are just a few of the possible shapes thatcan be uniformly heated. Other shapes, such as slices or cubes, are alsosuitable. Still other shapes that permit uniform and rapid heating areshapes in which heating elements—such as in the form of vanes—intersectthe sample. In other embodiments, the shaped sample has at least thefollowing attribute: its surface area to volume ratio is greater thanthat of a sphere of the same volume by a factor that is at least about2, and preferably at least about 3, 5, 10, 20, or 100.

Some types of biological samples, however, are either too small toshape, or are such that valuable information could be lost if part ofthe sample were removed. These biological samples 7 can be introducedinto a filler 9, which has a shape that allows for uniform heating ofthe sample, particularly when radiation heating is used. The filler 9preferably has a similar dielectric constant and electrical conductivityas the biological sample in order to facilitate the uniform heating, andis preferably inert. In some embodiments, the filler is a fluid or a gelthat includes water and salt ions. Alternatively, or in addition, forthe case of heating a sample by application of microwave radiation, thefiller has similar transmission characteristics with respect toelectromagnetic radiation, such as its refractive indices and absorptioncoefficients, to those of the biological sample, which thereby preventsuneven heating of the sample.

The parallelepipeds 1, 3 and right cylinder 5 are thin and oblong inrelation to their thickness, thereby facilitating rapid heat transfer incase of heat applied by contact with the sample. Examples of such heattransfer include conduction by gas, e.g., a flow of hot or warm gasdirected onto the sample, condensing gas, e.g., a flow of vaporizedliquid directed onto the sample, where the liquid has a low enoughboiling point that the liquid is able to form condensation on sample attemperatures below, for example, 200° C., or a warm plate contacting thebiological sample or contacting a container in which the biologicalsample is located.

The sample can be shaped by cutting the sample into the desired shape.In some embodiments, the sample is pressed, or flattened, such as byapplying pressure, to achieve the desired shape. In some embodiments,the sample has a thickness of between about 1 to 2,000 microns, such asbetween 1 and 1,000 microns, 1 and 500 microns, 1 and 200 microns, 1 and100 microns, 1 and 50 microns, 1 and 25 microns, 1 and 20 microns, 1 and10 microns or 1 and 5 microns. It is to be understood that the variousupper and lower endpoints of the foregoing ranges may be interchangedwith one another without limitation: for example, although notspecifically recited hereinabove, a range of 10-50 microns is alsoconsidered within the scope of the present invention, as is 500-1,000microns.

It is also to be understood that the term ‘about’ as used herein, inconnection with any quantity such as a time, or a length, or a mass, isintended to mean that the value in question may vary by up to 5% smalleror larger than the quoted value. Thus, for example, a thickness of about10 microns is intended to mean any thickness in the range 9.5 to 10.5microns. For temperatures, the term ‘about’ means that a variation of±2° C. is intended.

The methods of the present invention can be applied to both fluid andsolid samples. Samples that are solid may contain liquids within them(e.g., cells in tissues have liquid phase constituents within their cellwalls), but such samples have more solid characteristics than liquidcharacteristics, and thus can have a definite shape and volume and neednot be held by a container to maintain the shape and volume. Tissues,such as muscles, skin, brain, liver, kidney, bone, bone marrow, orothers can be considered to be solid samples. Fluid samples, whilepossibly having components that are more solid than fluid, primarilyhave fluid characteristics, such as the propensity to flow and to deformwhen very little external force is applied. Blood, blood components,urine, semen, CSF, lymph fluid, cell extracts, saliva and tears are someof the bodily fluids that can be analyzed using the techniques describedherein.

Fluid samples can be introduced into a container for retaining thesample during heating. The container has a shape that allows uniformheating of the fluid sample. In some embodiments, the fluid sample isintroduced into a tube or needle. The container can also be in one of aparallelepiped, a cylinder or other suitable shape, as shown in FIG. 3.Alternatively, a flat passage or a whirl canal can be used. The samplecontainer can also be configured for easy access to the sample afterheating, as the heating can in some instances change the sample fromhaving fluid characteristics to solid characteristics.

The sample container can be formed of a material that permits effectiveheat transfer through the container, such as a metal. The time forheating of the biological sample is therefore, in such instances, almostcompletely dependent on the heat conduction inside the sample since theheat conduction of the metal is far higher than that in the biologicalsample. For example, aluminium of common quality has a thermalconductivity of 190 W/mK, whereas a biological sample typically hasconductivity below 1 W/mK. The sample container can also be formed of amaterial that does not interfere with the transfer of radiation energyinto the sample, such as glass or other dielectric material or apolymer.

Unlike irradiation of a live organism where the organism is euthanizedby the irradiation process, and unlike irradiation of a part of anorganism such as the organism's head or its limbs, in the presentinvention the target sample is specifically shaped to ensure that thesample is uniformly heated and that all portions of the sample attainthe desired temperature at approximately the same time. Most organismsor parts of organisms are not ideally shaped for uniform heating byirradiation or any other method of applying heat.

Freezing the Sample

Optionally, the sample can be frozen, such as by flash freezing, priorto analysis. The sample can be brought to a temperature preferably below−20° C., such as −80° C. One advantage of freezing the sample is thatthe sample can be manipulated and shaped into the desired shape foruniform heating more easily when the sample is in a frozen state thanwhen fresh. Frozen samples can be cut into thickness of less than about5 mm, about 4 mm, about 3 mm, about 2 mm, about 1 mm or 0.5 mm.Preferably, for a frozen sample, the sample shape is a thin sheet, onthe order of microns thick. Such thin slices are less easily achievedwhen cutting a fresh sample. The freezing solidifies any liquid in thesample, including in samples considered to be solid, and allows for moreprecise cutting of the sample. Additionally, freezing arrestsproteolytic activity and prevents degradation of other components of thesample.

When the sample is frozen after being extracted from the organism, thesample should be kept frozen, such as below −20° C. or below −4° C.prior to heating. When a sample is frozen, ice crystals form and disturbthe plasma membrane. In addition, as a frozen sample thaws, vesiclemembranes become permeable. Increased permeability can cause proteindegradation to occur more rapidly once the sample is thawed than insamples that have never been frozen. With some types of biologicalsamples, the sample is not permitted to thaw before applying heat to thesample. That is, the sample is not allowed come to a temperature above−20° C. before the heat is applied. If the sample is thawed, the sampleis heated within about 30 seconds from the thawing of the sample, toprevent massive degradation from occurring.

Heating

After the sample has been shaped, the sample is then heated uniformly toa temperature that denatures macromolecules in the sample withoutdegrading the primary structure of those and other macromolecules. Theheating can be carried out by heat transfer from conduction, convection,or radiation. Additionally and alternatively, heating of the sample canbe accomplished by directing microwave radiation on to the sample.

The sample can be heated to about 55, 60, 70, 80, 90, 95, or 100° C. atnormal pressure, or the boiling point of a fluid sample, depending onthe type of molecule that is to be denatured. In some embodiments, thesample is prevented from being raised over a threshold temperature, suchas the boiling point of the sample, or 100° C. at normal pressure, sothat the primary structure is not destroyed. The sample can be heated ata higher temperature under pressure to denature the macromolecules.Maintaining the temperature of the sample below a threshold, and therebymaintaining the macrostructure, can facilitate sample analysis. If thetemperature achieved by the heating step causes the sample to reach atemperature at which the secondary structure of a macromolecule isdisrupted, the macromolecule is denatured. In certain instances, theheating disables proteolytic enzymes that break other proteins andpolypeptides into separate peptide fragments. The heating can arrest atleast 60%, such as at least 70%, 80%, 90% or 95% of the proteolyticactivity of the sample. The heating can also alter the tertiary andsecondary structure of the proteins and polypeptides of interest.However, the heating does not degrade the primary structures ofmacromolecules, and disables other molecules within the sample thatwould degrade primary structure, as well.

Any of the heating devices can heat the sample rapidly, such as in lessthan 2 minutes, less than 1 minute, less than 30 seconds, less than 10seconds, less than 5 seconds, less than 2 seconds, or less than 1second. In some embodiments, the heating brings all parts of the sampleto at least 60° C. within 2-3 minutes. Heating devices are describedherein that work using conduction or radiation. Conduction heating canbe used in instances where radiation will not heat a sample uniformly.In a sample that is frozen, using microwave radiation heating can causesome parts of the sample to attain the desired denaturation temperaturebefore other parts of the sample. By way of analogy, a block of iceheated in the microwave will resist thawing, because the hydrogen bondedmolecular network is not altered by the microwaves. For example, ice isthawed more efficiently by conduction than by applying microwaveradiation. As the ice begins to melt in some areas, the melted ice,i.e., the water, begins to warm up and heats the surrounding ice byconduction. This can allow some parts of the ice block to thaw and reachboiling prior to other parts of the ice block thawing. This phenomenonin a biological sample causes uneven heating, which can allow for morepeptide fragments to be present in the sample than would be present in asample that is uniformly heated to the target temperature. One optionfor avoiding this is to thaw the sample prior to the heating step.Another option is to use a heating method other than microwave radiationfor heating frozen samples. Such heating steps can avoid a separatethawing step altogether.

Timing

There are two phases after sample extraction from the organism in whichthe sample can degrade. The first phase begins at extraction and ends atthe initiation of the heating step. The second phase beginning at theinitiation of the heating step and ends when the sample reaches thedesired temperature. If the sample is not frozen, the combination of thefirst and second phases should be completed prior to the sampledegradation, i.e., prior to ion imbalance or depletion of ATP level andsubsequent increased levels of molecular fragments in the sample.Described herein are methods for determining the time that applies foreach type of biological sample. However, for a never-frozen sample it isdesirable that the two phases are completed within 10 minutes, such aswithin 5 minutes, 3 minutes, 2 minutes, 1 minute, or 30 seconds. In someimplementations, the second phase occurs within 2 minutes, 1 minute, 30seconds, 20 seconds, 10 seconds, 5 seconds, 2 seconds, 1 second or less.The first phase can be both extended and shortened if the sample isfrozen. The phase is extended, because the sample can be kept frozen forextended periods of time, such as days, weeks, months, or even years.However, the first phase is shortened in that the time between thawingthe sample and heating the sample must be kept very short because of theacceleration of degradation that occurs after the sample is thawed.

Examples of Biological Analysis

After the samples are heated to a suitable temperature for denaturingthe molecules, the samples can be analyzed to determine the protein andpolypeptide make up of the sample. The samples can be analyzed usingchromatography, mass spectroscopy, Edman degradation, or immunoassaymethods such as immunostaining, immunoprecipitation, western blot,enzyme assays, or other suitable analysis methods.

Exemplary Devices

Regardless of whether the sample is fresh or frozen when heated, thesample can be placed in a container that can be evacuated so that thesample can contact a heat source directly, without a pocket of airbetween the sample and the heat source. The container can be adeformable piece of material, such as a bag or a foil, that does notrelease molecules that would interfere with analysis results. Suitablematerials for such a container can include polymers, such as medicalgrade polymers, or other materials that do not give off gas, or havecomponents that can migrate into the sample during sample handling.

The prepared samples are heated in a device that is configured foruniformly heating the sample. Referring to FIG. 4, an embodiment of aheating device 11 is shown. The heating device 11 has a chamber 13 forreceiving a biological sample or a container that holds the sample, anda heating element 15 for heating the sample. An opening 17 allows thebiological sample (optionally in a container) to enter the chamber 13.The biological sample (optionally in a container) is introduced into thechamber 13 via the opening 17, and an inner wall 19 of the chamber 13contacts the biological sample, or a container that holds it. Thechamber 13 presents a large heat transfer surface in relation to itsvolume and is very close to the sample. The inner wall 19 is formed of amaterial that is capable of conducting heat to the sample. A thermallyinsulating layer 21 is provided around the chamber 13, to retain theheat within the chamber 13 and to protect objects outside of the devicefrom being heated. A heat sensor 23 and heat controller 25 are providedfor controlling the heating element 15. The heat controller 25optionally has a timer to regulate the on time of the heating element15, or to regulate the amount of time that heat is delivered to thesample. The device 11 can be powered by a battery or by electricity froma power supply, connected by an electrical cord (not shown). In thisembodiment, the biological sample may be either fluid, for exampleconfined to a container, or solid.

Referring to FIG. 5, another embodiment of a device 11 is configured forallowing the sample to enter and exit through separate openings. Thesame reference numerals are used in FIGS. 4 and 5 to refer to the samefeatures. Device 11 in FIG. 5 has a second opening 33 to allow a fluidbiological sample to flow out of the chamber 13. A biological samplethat can be used with the heating device of FIG. 5 is preferably a fluidwhich is non-coagulating. The biological sample is physically shaped asit is introduced into the chamber 13. In this embodiment, the crosssection of the chamber 13 is designed so that it presents a wide baseand a low height, with the intention of a fast and uniform heat transferthrough the whole biological sample.

FIG. 6 shows an alternative embodiment of a device 41, configured foruse with a sample that is introduced into the heating chamber in acontainer. The biological sample in a container 43, such as a tube or aneedle, is introduced into a chamber 13 via the opening 17. The opening17 has an O-ring 45 (shown in cross-section in FIG. 6), which seals thecontainer 43 into the chamber 13. The chamber 13 and the container 43are sized so that the container 43 does not contact all of the walls ofthe chamber 13, and in some embodiments, the container 43 contactsnothing other than the O-ring 45. This arrangement provides a space inwhich steam can be introduced for heating the sample. A steam generator49 communicates with the chamber 13 through a steam inlet 47. Thechamber 13 optionally includes a steam pressure control (not shown). Assteam enters the chamber 13, the container 43 and the biological samplewithin it (not shown) are heated. In some embodiments, the steam isreplaced by another heated fluid.

Referring to FIG. 7, a plan view of a heating device 51 in which samplesin a container can be introduced is shown. The device's chamber 13comprises two moveable matching parts 53, 55 and a locking means 57 formaintaining the two matching parts 53, 55 in a locked state. The twomoveable matching parts 53, 55 can rotate apart, such as by rotatingaround hinge 57. The device 51 according to this embodiment is openedand closed as indicated by the arrows. Alternatively, the moveable partscan slide open along a track. A biological sample in a container 43,such as a tube or a needle, is put into the chamber 13 when the moveablematching parts 53, 55 are moved so that the device is open. Afterclosing and locking the two matching parts 53, 55, the heating controlmeans 25 is activated. Alternatively, the heating control means 25 isactivated prior to introducing the sample into the heating device 51. Athermally insulating layer 21 is provided around the chamber 13. Aninner wall 19 of the chamber 13 is in contact with the container 43.This allows fast and uniform heat transfer through the whole biologicalsample. Although the device 51 is shown with two moveable pieces, othernumbers of moveable pieces can be incorporated into the device,consistent with the foregoing description.

In some embodiments, the heat source is a single plate heated, e.g., bya heating element, and the sample, regardless of whether it is placed ina container, receives heat from one side only, i.e., there is a singlecontact surface allowing power to be transferred into the biologicalsample.

Referring to FIG. 8, a device 61 that uses radiation (e.g., microwaveradiation) to heat the biological sample is shown. The device 61comprises a chamber 13 for receiving the biological sample 7. The sourceof radiation 15 can be a microwave generator. Alternatively, other typesof radiation can be applied to the sample, such as radiofrequency (RF)or ultrasound. This embodiment also comprises an opening 17, which canbe covered by a door, for allowing the biological sample 7 to enter thechamber 13. The biological sample 7 is introduced into the chamber 13via the opening 17. A heat sensor 23 and heat controller 25 are providedfor controlling the heater 15. The source of radiation can output about1 to 6 Watts of energy, such as between about 3 and 5 watts. In someembodiments, between about 2 and 4 Watts/minute/gram, such as 3.6Watts*minute, are input into the sample to raise the temperature of thesample from 20 to 80 degrees, if the biological sample has a thermalcapacity similar to water. The needed radiation energy is 3.6/efficencyWatts*minutes/gram., i.e., if the efficiency is 10%, the neededradiation is 36 Watts*minutes/gram. The mass includes both the sampleand the filler, i.e., if the filler has a similar mass to the sample andthe sample weighs 10 grams, with an efficiency of 10% then 360 Watts areneeded to heat the sample to 80 degrees in one minute. Thus, about 72Watts are required to heat one gram at 20° C. to 80° C. in 30 seconds.

The chambers described in any of the devices mentioned herein can haveother shapes including, but not limited to, a rectangular, square,circular, oval or triangular cross section.

Referring to FIG. 9, a device for conductively heating a sample isshown. The device 81 has two retaining members 85 that are capable ofconducting heat from one or more respective heat sources. In someembodiments, the retaining members are formed of a material having highthermal conductivity, such as a metal. It need not be necessary tocontinually supply heat to the retaining members; this is particularlyso if the members are sufficiently large that the temperature does notdrop on the plates by more than 10° C. when the sample is added. In someembodiments, the members 85 have a layer of gel or oil that is heatedand covered by a protecting layer, such as a plastic. The gel or oil isdeformable and is able to contact all parts of the biological sampleduring heating. Optionally, the members have a recessed area forretaining the sample. The members can completely surround the sample. Aseal 87 can be provided around the recessed area on the members 85 inwhich the sample is placed, so that when the members are broughttogether, a vacuum can be applied to the sample to eliminate air pocketsbetween the sample and the members 85. The vacuum can be applied by avacuum pump (not shown). In one embodiment, to create a vacuum, a lumen(not shown) is formed between the sample holding portion of at least onemember 85 and a vacuum source. The members are connected to an energysource, such as electricity, so that the members can be heated. Themembers can have substantial mass in comparison to the sample so thatwhen the sample is placed on the members, a negligible change intemperature is experienced by the members 85.

During operation of device 81, the sample can be protected from themembers by a layer of material that can endure heating, but which doesnot release volatile molecules when heated, such as silicone,polycarbonate or PTFE (e.g., TEFLON™). A sensor can sense thetemperature of the members 85 and ensure that the temperature does notbecome too high. The sensor can be an IR camera or a thermocouple orother suitable sensor. The sensor, or sensors, can be connected to oneor both of the members 85. The members 85 can be moved closer togetherand further apart (in the directions of arrows) to allow for loading andretrieving of the sample. Optionally, a transfer element 89 can be usedto load and retrieve the sample. The transfer element 89 can be within achilled environment. In some embodiments, the chilled environment isdefined by a housing (not shown) surrounding a portion of the device 81.A source of chilled air can be supplied within the housing to maintain adesired temperature. In some embodiments, the transfer element 89 andapparatus surrounding the transfer element 89 are themselves chilled,such as by a source of chilled fluid. The chilled element 89 and/orenvironment can maintain the sample in a frozen state, preferably below−20° C., prior to heating.

Example 1 Analysis of Mouse Brain Samples

Twelve mice (C57/BL6) were euthanized by cervical dislocation. Thetwelve mice were divided into three groups and each mouse brain was keptat room temperature (22° C.) for 1, 3, or 10 minutes. At theirrespective time point, the mouse brains were irradiated in a smallanimal microwave for 1.4 seconds at 4.5-5 kW. A fourth group waseuthanized by focused microwave irradiation (the control group). Thestriatum, hypothalamus and cortex were thereafter rapidly dissected outof all of the mice after microwave radiation and stored at −80° C.

An additional group of four mice were also euthanized by cervicaldislocation and the mouse heads were immediately cooled in liquidnitrogen. The striatum was rapidly dissected out on dry ice and frozenat −80° C. The frozen samples were shaped into thin slices. Half of thegroup of samples were then immediately heated to near 100° C. using acontact heating device. The other half of the samples were thawed andprepared at 4° C.

The brain tissues were then suspended in cold extraction solution (0.25%acetic acid) and homogenized by microtip sonication (Vibra cell 750,Sonics & Materials Inc., Newtown, Conn.) to a concentration of 0.2 mgtissue/μL. The suspension was centrifuged at 20,000 g for 30 min at 4°C. The protein- and peptide-containing supernatant was transferred to acentrifugal filter device (Microcon YM-10, Millipore, Bedford, Mass.)with a nominal molecular weight limit of 10,000 Da, and centrifuged at14,000 g for 45 min at 4° C. Finally, the peptide filtrate was frozenand stored at −80° C. until analysis.

The samples were then analyzed, by monitoring phosphorylated proteins byphosphor-specific western blotting and by qualitative and quantitativepeptide analysis, using nanoLC/ESI MS.

The samples that were allowed to remain at room temperature aftercervical dislocation were compared to the control group. The controlgroup displayed an average of 660±70 distinct MS peaks from striatumusing nanoLC ESI MS. Within one minute post mortem, protein fragmentsfrom degrading proteins were detected, and the peptides increased to1060±400 peaks. Three minutes post mortem the number peptides reached2150±800 and after 10 minutes, the number of peptides reached 2800±500.

The samples that were in vitro inactivated on snap frozen samples werecompared to non-inactivated samples (non-heated samples) and the controlsamples. In the samples that were merely frozen, allowed to thaw to 4°C. and prepared, a large number of peptides were detected. The samplesthat were heat treated immediately after being dissected on icedemonstrated peptide identities analogous to the control results.

The techniques described herein can offer a number of advantages. Lowabundance peptides, such as neuropeptides, remain intact for analysis,proteolytic degradation of proteins is minimized and post-translationalmodifications of the neuropeptides are conserved. The samples can befixed without the use of fixing solutions, such as aldehydes. Whensamples free of fixation solution are analyzed, there are fewerimpurities that must be factored out from the data. While fixingsolutions can maintain the physical shape of tissues, the fixingsolution can change the molecules. The heating process is able to fixthe samples and maintain information that is lost when a fixing solutionis used. The techniques described herein allow the samples to beanalyzed for detection of abnormalities, such as disease markers.Additionally, autopsy samples, bio bank samples, biopsy samples andother samples that are not suitable for in vivo inactivation ordenaturation can be sampled without loss of polypeptide information.Samples removed at a clinic and frozen can be subsequently analyzedusing technique described herein to diagnose diseases or abnormalities.

The foregoing description is intended to illustrate various aspects ofthe present invention. It is not intended that the examples presentedherein limit the scope of the present invention. The invention now beingfully described, it will be apparent to one of ordinary skill in the artthat many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

All publications referred to herein are hereby incorporated by referencefor all purposes.

What is claimed is:
 1. A method for preparing a biological sample foranalysis, the sample comprising at least one macromolecule having aprimary structure and a secondary structure, the method comprising:forming a shaped sample by causing a volume of the sample to adopt ashape wherein its surface area to volume ratio is greater than that of asphere of the same volume by a factor that is at least about 2 and athickness between about 1 to 2000 microns so as to permit uniform andrapid heating; and uniformly heating the shaped sample by heatconduction using corresponding conductive elements of specified shapesso that the secondary structure of the macromolecule is disrupted, butthe primary structure is not, wherein, during the heating, the sample issubstantially free of aldehydes or fixative material.
 2. The method ofclaim 1, wherein the method further comprises: extracting the biologicalsample from an organism.
 3. The method of claim 2, wherein the organismis a human.
 4. The method of claim 2, wherein the organism is an animal.5. The method of claim 1, wherein the causing takes place at a firstperiod of time after extracting the biological sample from an organism,wherein the first period of time is sufficiently short to preventdegradation of the primary structure of the macromolecule.
 6. The methodof claim 5, wherein the first period of time is 3 minutes.
 7. The methodof claim 5, wherein the first period of time is 2 minutes.
 8. The methodof claim 1, wherein the heating is carried out for a second period oftime, wherein the second period of time is from about 10 seconds toabout 2 minutes.
 9. The method of claim 1, wherein the second period oftime is up to about 10 seconds.
 10. The method of claim 1, wherein theheating includes heating the shaped sample to at least 70° C.
 11. Themethod of claim 1, wherein the sample is a tissue predominantly in solidphase.
 12. The method of claim 1, wherein the sample is predominantly inliquid phase.
 13. The method of claim 1, wherein the causing comprisespressing the biological sample into a thin film.
 14. The method of claim1, wherein the shaped sample is a slice that is between about 1 andabout 1,000 microns thick.
 15. The method of claim 1, wherein the shapedsample is such that no part of it is greater than a threshold distancefrom a source of heating, and wherein the threshold distance is between1 mm and 1 cm.
 16. The method of claim 1, wherein the causing comprisesadding a filler to the biological sample.
 17. The method of claim 16,wherein the filler has a similar dielectric constant to a dielectricconstant of the biological sample.
 18. The method of claim 1, furthercomprising freezing the sample prior to the heating.
 19. The method ofclaim 18, wherein the freezing comprises reducing the sample to atemperature below −20° C. immediately after extraction from an organism.20. The method of claim 18, wherein the heating is initiated while theshaped sample is below −20° C.
 21. The method of claim 18, wherein theheating is initiated when the shaped sample is above −20° C. for asufficiently short time to prevent degradation of the primary structureof the macromolecule.
 22. The method of claim 1, wherein the causingincludes placing the biological sample in a container, reducing thepressure within the container, and sealing the container after reducingthe pressure.
 23. The method of claim 1, wherein the sample is heated ina system comprising: a heat source; a retaining member in communicationwith the heat source, and configured to contact the biological sample,and wherein the retaining member conducts heat from the heat source intothe biological sample; a zone in which the biological sample can be heldat a controlled temperature; and a transfer element configured to movethe biological sample out from the zone and onto the retaining member.24. The method of claim 1, wherein the sample is heated in a heatingdevice comprising: a chamber configured to receive a shaped biologicalsample, wherein the chamber has one or more internal surfaces that arein contact with the biological sample, and wherein the sample is totallycontained within the chamber; one or more heating elements incommunication with the one or more internal surfaces; a heat sensor incommunication with the one or more internal surfaces; and an inletadapted to permit the sample to be directed into the chamber; andwherein the chamber has a shape so that no part of the sample is greaterthan 10 mm from a point on any one of the one or more internal surfaces.25. The method of claim 1, wherein the sample is not heated by microwaveirradiation.
 26. A method for preparing a biological sample foranalysis, the sample comprising at least one macromolecule having aprimary structure and a secondary structure, the method comprising:forming a shaped sample by causing a volume of the sample to adopt ashape wherein its surface area to volume ratio is greater than that of asphere of the same volume by a factor that is at least about 2 and athickness between about 1 to 2000 microns so as to permit uniform andrapid heating; and uniformly heating the shaped sample by heatconduction using corresponding conductive elements of specified shapesso that the secondary structure of the macromolecule is disrupted, butthe primary structure is not, wherein the heating reduces proteolyticactivity by at least 70%, wherein, during the heating, the sample issubstantially free of aldehydes or fixative material.
 27. The method ofclaim 26, wherein the heating reduces proteolytic activity by at least80%.
 28. A method for preparing a biological sample for analysis, thesample comprising a proteolytic molecule and a polypeptide, wherein thepolypeptide has a primary structure and secondary structure, the methodcomprising: forming a shaped biological sample by causing a volume ofthe sample to adopt a shape wherein its surface area to volume ratio isgreater than that of a sphere of the same volume by a factor that is atleast about 2 and a thickness between about 1 to 2000 microns so as topermit uniform and rapid heating; uniformly heating the biologicalsample by heat conduction using corresponding conductive elements ofspecified shapes to cause the sample to uniformly attain a temperatureat which the activity of the proteolytic molecule is disrupted enough sothat the proteolytic molecule is unable to degrade the primary structureof the polypeptide, wherein, during the heating, the sample issubstantially free of aldehydes or fixative material.
 29. The method ofclaim 28, wherein the heating occurs before the proteolytic molecule hasreduced the concentration of the polypeptide by 50% relative to asimilar sample in a living organism.
 30. A method for preparing abiological sample from an organism for analysis, the sample comprisingat least one macromolecule of interest, wherein the macromolecule ofinterest has a primary structure and a secondary structure, the methodcomprising: forming a shaped sample by causing a volume of the sample toadopt a shape wherein its surface area to volume ratio is greater thanthat of a sphere of the same volume by a factor that is at least about 2and a thickness between about 1 to 2000 microns so as to permit uniformand rapid heating; and uniformly heating the shaped sample by heatconduction corresponding conductive elements of specified shapes tocause the shaped sample to attain a temperature wherein the temperaturecauses a secondary structure of a digestive molecule to degrade, whereinthe digestive molecule naturally digests the macromolecule of interestwhen its secondary structure is intact, wherein the temperature does notcause the primary structure of the macromolecule of interest to degrade,and wherein, during the heating, the sample is substantially free ofaldehydes or fixative material.